The present invention relates to a novel restriction endonuclease, Bcg I, obtainable from Bacillus coagulans, to the process for producing the same, and to related methods of employing this novel enzyme.
Many bacteria contain systems which guard against invasion of foreign DNA. Bacterial cells contain specific endonucleases that make double-strand scissions in invading DNA unless the DNA has been previously modified, usually by the appropriate corresponding DNA methylase. The endonuclease with its accompanying methylase is called a restriction-modification system (hereinafter "R-M system"). The principle function of R-M systems is thus defensive: they enable bacterial cells to resist infections by bacteriophage and plasmid DNA molecules which might otherwise parasitize them.
Bacteria usually possess only a small number of restriction endonucleases per species. The endonucleases are named according to the bacteria from which they are derived. Thus, the species Haemophilus aegyptius, for example synthesizes three different restriction endonucleases, named Hae I, Hae II, and Hae III. These enzymes recognize and cleave the sequences (AT)GGCC(AT), PuGCGCPy and GGCC respectively. Escherichia coli RY13, on the other hand, synthesizes only one restriction endonuclease, EcoR I, which recognizes the sequence GAATTC.
Restriction endonucleases, the first component of R-M systems, have been characterized primarily with respect to their recognition sequence and cleavage specificity because of their practical use for molecular dissection of DNA. The majority of restriction endonucleases recognize sequences 4-6 nucleotides in length. More recently, recognition endonucleases having recognition sequences of 7-8 nucleotides in length have been found. Most, but not all, recognition sites contain a dyad axis of symmetry, and in most cases, all the bases within the site are uniquely specified. This symmetrical relationship in the recognition sequence of restriction endonucleases has been termed "palindromes". Some restriction endonucleases have degenerate or relaxed specificities in that they can recognize multiple bases at the same positions. Hae III, which recognizes the sequence GGCC is an example of restriction endonuclease having a symmetrical relationship, while Hae II, which recognizes the sequence PuGCGCPy, typifies restriction endonucleases having a degenerate or relaxed specificity. Endonucleases with symmetrical recognition sites generally cleave symmetrically within or adjacent to the recognition site, while those that recognize asymmetric sites tend to cut at distance from the recognition site, typically from about 1-18 base pairs away from the site.
The second component of bacterial R-M system is the modification methylase. These enzymes are complementary to restriction endonucleases and provide the means by which bacteria are able to protect their own DNA and distinguish it from foreign, infecting DNA. Modification methylases recognize and bind to the same nucleotide recognition sequence as the corresponding restriction endonuclease, but instead of breaking the DNA, they chemically modify one or other of the nucleotides within the sequence by the addition of a methyl group. Following methylation, the recognition sequence is no longer bound or cleaved by the corresponding restriction endonuclease. The DNA of a bacterial cell is always fully modified, by virtue of the activity of its modification methylase and it is therefore completely insensitive to the presence of the endogenous restriction endonuclease. It is only unmodified, and therefore identifiably foreign, DNA that is sensitive to restriction endonuclease recognition and attack.
More than 1,000 different restriction endonucleases have been isolated from bacterial strains, and many share common specificities. Restriction endonucleases which recognize identical sequences are called "isoschizomers". Although the recognition sequences of isoschizomers are the same, they may vary with respect to site of cleavage (e.g., XmaI, SmaI Endow, et al., J. Mol. Biol. 112:521 (1977); Waalwijk, et al., Nucleic Acids Res. 5:3231 (1978)) and in cleavage rate at various sites (XhoI v. Pae R7I Gingeras, et al., Proc. Natl. Acad. Sci U.S.A. 80:402 (1983)).
Three distinct types of R-M systems have been characterized on the basis of the subunit compositions, co-factor requirements, and type of DNA cleavage. Type I R-M systems are the most complex. The endonuclease typically contains three different types of subunits and require Mg.sup.++, ATP, and S-adenosyl-methionine for DNA cleavage. Their recognition sites are complex, and DNA cleavage occurs at non-specific sites anywhere from 400-7,000 base pairs from the recognition site.
Type II R-M systems are much simpler than either types I or III. The endonuclease only contains one subunit, and only Mg.sup.++ is required for DNA cleavage. Moreover, the DNA cleavage site occurs within or adjacent to the enzymes' recognition site. It is this class of restriction endonucleases that has proved most useful to molecular biologists.
Type III R-M systems are somewhat less complex than type I systems. The endonuclease of type III R-M systems contain only two types of subunits, and although Mg.sup.++ and ATP are required for DNA cleavage, S-adenosyl-methionine stimulates enzymatic activity without being an absolute requirement. DNA cleavage occurs distal to the recognition site by about 25-27 base pairs.
Recently, two restriction endonucleases, Gsu I and Eco57 I, were isolated and identified. Both require Mg.sup.++ and are stimulated by S-adenosyl-methionine (see: Petrusyte et al., Dokl. Akad. Nauk. SSSR 295 p.1250-1253, 1987). These enzymes appear to be a new type of restriction endonuclease. Furthermore, Kramarov, et al. Bioorg, Khim 14 p.916-920, 1980, reported site specific endonuclease BST 4.4I from Bacillus stearothermophilus which was reported to produce two double strand cuts separated with 30 to 32 nucleotides in the region of the recognition site. Additionally, Orekhov et al., Dokl. Akad. Nauk. SSSR 263 p.217-220, 1982 reported Sgr II, an isoschizomer of EcoRI I from Streptomyces griseus, and suggests that it cleaves on both sides of its recognition site.
While a large number of restriction endonucleases are already known for numerous DNA sequences, there is a continuing need for restriction enzymes with diversified enzymatic characteristics for successful genetic manipulation.